EVALUATION OF SERUM MALONDIALDEHYDE SPECTROPHOTOMETRICALLY AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY AND ITS RELATIONSHIP WITH CORONARY ARTRY DISEASE

Atherosclerosis and coronary artery disease are the leading causes of death in developed and also developing countries. Reactive species, especially those derived from oxygen, are mediators of vascular and tissue damage in various diseases including coronary artery disease. Determination of this reactive species by direct method is expensive, but we can determine the product of this reactive species indirectly i.e. MDA. MDA level is determined by thiobarbituric acid reactive substances (TBARS) and HPLC methods. The main purpose of this study was to compare and evaluate HPLC and TBARS methods for determination of serum MDA in a group of patients with coronary artery disease in comparison to control subjects.

Study populations were 47 controls and 53 patients with coronary artery disease. Blood samples were collected after an overnight fasting, and sera were separated by centrifugation. For MDA determination, first serum protein was precipitated by trichloroacetic acid, and separated by centrifugation, and supernatant was reacted with thiobarbituric acid solution in 950C for 50 minutes. For determination of TBARS, TBA-MDA adduct measured spectrophotometrically by reading of absorbance directly at 532 nm, and for determination of MDA, 20 µL of each reacted sample was injected to a C8 revesephase column of HPLC, separated isocratically, and quantitated by visible detector at 532 nm.

The recovery of HPLC and TBARS methods were 92.05 - 105.2 and 84.7 - 102 respectively. The precision of HPLC method was 4-6.17 while in TBARS method it was 7.27 - 12.22%. The detection limit of HPLC and TBARS method were 0.05 µM and 0.1 µM respectively. Serum levels of MDA which determined by TBARS method was higher than those determined by HPLC in the same samples (P value <0.002). There were significant correlations between the results of TBARS and HPLC method for serum MDA in all subjects. (r2 = 0.325, P value = 0.02).

However, HPLC is a sensitive and accurate method for determination of MDA, but it is time consuming and very expensive. So in improved and optimized conditions, the results of TBARS could be acceptable, and this method is suitable for routine laboratory use, and larger sample size epidemiological studies. According to our results and previous findings, serum levels of MDA in patient with coronary artery disease were higher than the control group, so it is an independent risk factor or a marker for cardiovascular diseases.